Chang M S, Chang G D, Leu J H, Huang F L, Chou C K, Huang C J, Lo T B
Department of Zoology, National Taiwan University, Taipei.
DNA Cell Biol. 1996 Oct;15(10):827-44. doi: 10.1089/dna.1996.15.827.
A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3, and human TYK2 is 57%, 35.5%, 31.3%, and 42.4%, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JH1) domains (approximately 70% identity). Therefore, carp JAK1 is a homolog of mammalian JAK1. To investigate the possible function of JH2 domain, full-length, and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JH1 and c-JH2 associate with each other and c-JH2 can be tyrosine-phosphorylated by c-JAK1 and by c-JH(1 + 2). The JAK1 gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. The 2.5-kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, AP1, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT-Basic. Deletion analysis defined a positive regulatory region between -1,023 and -528. A smaller region (-181 to +59) without any typical TATA-box sequences, G + C-rich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK1 gene.
一个3.7千碱基对的cDNA编码了由1156个氨基酸残基组成的鲤鱼JAK1激酶。鲤鱼JAK1与小鼠JAK1、JAK2、JAK3以及人类TYK2的整体氨基酸序列同一性分别为57%、35.5%、31.3%和42.4%。此外，鲤鱼JAK1在激酶样（JH2）和激酶（JH1）结构域中与哺乳动物JAK1显示出更高的序列同源性（约70%的同一性）。因此，鲤鱼JAK1是哺乳动物JAK1的同源物。为了研究JH2结构域的可能功能，在杆状病毒系统中产生了全长及各种截短形式的鲤鱼JAK1。我们的结果表明，c-JH1和c-JH2相互结合，并且c-JH2可以被c-JAK1和c-JH(1 + 2)酪氨酸磷酸化。JAK1基因也从鲤鱼基因组文库中分离出来并进行了表征。该基因被分为24个外显子，跨越至少31千碱基对的基因组DNA。外显子1包含5'-非翻译区，外显子2包含推定的翻译起始位点。转录起始位点上游2.5千碱基对的DNA区域包含众多转录因子的潜在结合位点，包括NF-IL6、HNF-5、AP1、GHF-5和E2A。当这个DNA片段置于氯霉素乙酰转移酶（CAT）报告基因上游并转染到鲤鱼CF细胞系中时，它驱动CAT酶合成的效率比无启动子的pCAT-Basic高16倍。缺失分析确定了-1023至-528之间的一个正调控区域。一个较小的区域（-181至+59），没有任何典型的TATA盒序列、富含G + C的序列或其他已知转录因子的结合序列，仍然具有启动子活性。没有这个区域的构建体没有可检测到的启动子活性。这表明该DNA区域可能在鲤鱼JAK1基因的表达中起重要作用。
